Protein folding is among the most important and most challenging problems in biophysics. Newly devised methods of characterizing high resolution structure in folding intermediates are pointing the way towards a solution. The approach is to investigate how know changes in the primary structure affect the kinetic and equilibrium properties of protein folding reactions. Methods include fast reaction techniques and NMR spectroscopy. Iso-1 and iso-2 cytochromes c are the major focus of the work since these proteins are particularly well suited for primary structure manipulations. Stopped flow mixing will be used to determine which of the kinetic processes in folding of the wild-type protein are perturbed by changes in the primary structure of the mutant proteins. The focus will be on proteins with mutations at or near conserved sites, especially prolines assigned to slow folding kinetic phases. Differences in stability will be measured for proteins with mutations located within or at the interface between elements. The time course of appearance of structure in native-like or molten globule folding intermediates is to be characterized. Monoclonal antibodies are used to determine the rate of appearance of specific native-like epitopes in refolding. Stopped-flow quench H-D pulse labeling and NMR spectroscopy are used to measure the time course of formation and the extent of H-bonded structure in early folding intermediates. The efficiency of catalysis of slow folding by prolyl isomerases (PPI and FKBP) is used to probe structure formation at or near specific proline residues. Tests are proposed of the relationship between mutation-induced changes in structure, local and global stability, and energetic constraints on side-chain motion. Differential scanning microcalorimetry is used to measure and compare the thermodynamic parameters of the thermal unfolding transitions for normal and mutant proteins with known high resolution X-ray structures. Rates of H-D exchange and aromatic ring flips are measured to probe local unfolding equilibria and the motion of side-chains, respectively. These studies of folding of mutant proteins will provide an understanding of the "feed back" mechanism by which long range interactions influence the choice between alternative local structures, and will aid in deciphering the code relating amino acid sequence to tertiary structure and function.